Analyte detection with a gradient lateral flow device

ABSTRACT

Methods for determining the concentration of an analyte in a sample in which an analyte gradient is established and brought into contact with one or more zones that contain binding members that interact with the analyte and thereby produce a detectable signal. Devices that may be used to practice the disclosed methods are also provided.

BACKGROUND OF THE INVENTION

The invention relates to analyte detection.

Lateral flow devices (LFDs) can be used outside the confines of aclinical or laboratory setting to detect the presence of variousanalytes in biological or other samples. Generally, these devices aresimple, disposable, and self-contained. A common feature of LFDs is anabsorbent sample application pad that draws the sample laterally, bycapillary action, across one or more reagents that interact with theanalyte (if it is present in the sample) and produce a measurablesignal. The principle underlying LFDs is the same as that underlyingother detection methods such as RIAs (radioimmunoassays) and ELISAs(enzyme-linked immunosorbant assays): the reagents in the LFD mustspecifically bind or otherwise interact with the analyte being tested.

Examples of absorptive-pad assay devices and methods are described inU.S. Pat. Nos. 3,983,005, 4,069,017, 4,144,306, 4,366,241, and5,354,692.

SUMMARY OF THE INVENTION

The invention features methods and devices for determining theconcentration of an analyte in a sample by establishing an analyteconcentration gradient and bringing that gradient into operable contactwith one or more zones within a novel lateral flow device. These zonesinclude, as described below, an indicator zone, a test zone, and acontrol zone. Devices that can be used to perform each of the methodsdescribed herein are also features of the invention.

In one embodiment, the concentration of an analyte in a sample isdetermined by establishing an analyte gradient, bringing that gradientinto operable contact with an indicator zone that contains a mobilebinding member, and then bringing the indicator zone into operablecontact with a test zone that contains a fixed binding member.

In a second embodiment, the concentration of an analyte in a sample isdetermined by establishing an analyte gradient and bringing thatgradient into operable contact with a test zone that contains a fixedbinding member.

In a third embodiment, the concentration of an analyte in a sample isdetermined by establishing an analyte gradient, bringing that gradientinto operable contact with a test zone containing a fixed bindingmember, and then bringing an indicator zone that contains a mobilebinding member, into operable contact with the test zone.

In each embodiment, a detectable signal, which indicates theconcentration of the analyte in the sample, is produced in the testzone.

The methods and devices described herein can be used in the context ofbasic scientific research, the practice of medicine, includingveterinary and dental medicine, forensic analysis, environmentalprotection studies, industrial or chemical manufacturing, and thedevelopment and testing of pharmaceutical, food, and cosmetic products.

The analyte detected may be, for example, a biological substance such asa hormone, an enzyme, an immunoglobulin, a bacterial, viral, parasitic,or fungal antigen, or the like. Alternatively, the analyte may be a drugthat is administered in the course of treating a disease or a drug ofabuse such as a barbiturate, an amphetamine, methadone, morphine,cocaine, codeine, dilaudid, tetrahydrocannabinol, or diazepam. Otherdetectable analytes include those generated by the manufacture of food,industrial agents, or chemical products. Examples of such analytesinclude food additives (e.g. bulking agents, vitamins, colorants orflavorants), agrichemicals (such as pesticides, insecticides,herbicides, and fertilizers), surfactants (e.g., sodium dodecylsulfate),adhesives (e.g. isocyanate glues), resins (e.g. wood resins and epoxyresins), organic pollutants (e.g. dioxins), and process chemicals (e.g.chemicals used in water systems) such as flocculating polymers,biocides, corrosion inhibitors, and anti-scalants. In addition, theanalyte can be a substance that is used for the purpose of marking ortracing a product or process.

Any liquid suspected of containing a specific analyte can be used as asample. These liquids include physiological fluids such as whole blood,plasma, serum, urine, cerebrospinal fluid, ascites fluid, sweat, lymph,or other body fluids. The antigens or antibodies present in these fluidsmay be monitored in order to determine the severity of an infection orto gauge the progress of treatment. For example, tumor antigens shedinto the bloodstream may be monitored following chemotherapy orradiation therapy.

Liquids obtained from manufacturing processes as well as liquidscollected from the environment may also be analyzed by the inventiondescribed herein. For example, rainwater, or water from an ocean, river,lake, pond, or stream may be collected and analyzed.

The sample may be processed, if necessary, prior to analysis. Forexample, the sample may be centrifuged or filtered to remove particulatematter or buffered in order to allow more efficient detection of theanalyte. Suitable buffers include any of those known to skilledartisans, such as a 1-1,000 mM solution of Tris (TRIZMA, Sigma ChemicalCo., St. Louis, Mo.) or 1-1,000 mM TRIS (2-Amino-2-(hydroxy-methyl)-1,3-propanediol). Other buffers include phosphate buffered saline (PBS),citrate buffer, or bicarbonate buffer.

Preferably, the methods and devices described herein will be used todetect 0,001-1×10⁷ μg/l, more preferably, 0.1-1×10⁵ μg/l, and mostpreferably 1-1×10⁴ μg/l of analyte.

The invention may also be used to detect analytes that are initiallycontained within solid-phase samples. These analytes would simply beextracted and suspended or dissolved in liquid prior to analysis. Theextraction process could be as simple as shaking a solid sample in abuffer such as those listed above, which could then be applied to thegradient lateral flow device. Solid samples may include biologicaltissues (obtained, for example, in the process of performing a biopsy),soil, or foliage.

The analyte gradient may be established in numerous ways. For example,the sample can be applied to a wedge-shaped sample application pad, thediluent can be applied to a wedge-shaped diluent application pad, withthe gradient being established by bringing the sample and diluentapplication pads into contact with one another. These pads may consistof an absorbent material, examples of which are given below. Although itis expected that the analyte gradient will be established with sample-and diluent application pads or chambers that are both wedge-shaped, agradient may be established by applying either the sample to awedge-shaped pad and the diluent to a square or rectangular-shaped pad,and vice-versa. The sample- and diluent application pads or chambers canbe designed to create a linear or non-linear analyte concentrationgradient.

The edges of the sample and diluent application pads may be smooth, orone or more of their edges may be stepped, as stairs are stepped. Thehorizontal and vertical aspects of each step may be the same length, orone aspect may be longer than the other. Similarly, all of the steps maybe identical, or they may differ. For example, the steps at theperiphery of the gradient may be larger than the steps in the center ofthe gradient.

Alternatively, the analyte gradient may be produced by applying thesample to a wedge-shaped sample application chamber, applying a diluentto a wedge-shaped diluent application chamber, and bringing the contentsof these two chambers into contact with one another. The chambers may beconstructed from any material that is capable of forming and retaining ashape. Examples of such materials include plastics, plexiglass, andglass. The chambers may be shaped to create either a smooth or a steppedanalyte gradient, as described above. To establish a stepped gradient,the sample-containing chamber and/or the diluent-containing chamber maybe subdivided into a parallel array of wells that end squarely where thesample- and diluent-containing chambers are brought into contact withone another. The wells could be manufactured, for example, by adjoininga series of capillaries of varying height.

The analyte gradient will move, as further described below, into contactwith either a mobile binding member in the indicator zone or a fixedbinding member in the test zone. For example, in the first embodiment,the analyte gradient contacts the indicator zone, wherein the analyteassociates with a mobile binding member. Subsequently, the mobilebinding member, or the analyte associated therewith, comes into contactwith the test zone and associates with a fixed binding member therein.Alternatively, the mobile binding member may be associated with ananalogue of the analyte that has a different, preferably lower, bindingaffinity for the mobile binding member than does the analyte. In thisinstance, the analyte would displace the analogue, which maysubsequently associate with the fixed binding member in the test zone.In the second embodiment, the analyte gradient contacts the test zonedirectly and associates with a fixed binding member therein. In thethird embodiment, the analyte gradient first contacts the test zone andassociates with a fixed binding member therein. Subsequently, theindicator zone is brought into contact with the test zone and a mobilebinding member (from the indicator zone) associates with either thefixed binding member in the test zone or the analyte associatedtherewith.

The binding member in the indicator zone is mobile (i.e., able to moveout of that zone), whereas the binding member in the test zone is fixed(i.e., unable to move out of that zone). Otherwise, the binding membersare equivalent and may include any substance that is capable of bindingto, or otherwise specifically interacting with, either the analyte oranother binding member that is associated with the analyte. Examples ofbinding members include antibodies, or a fragments thereof, antigens,haptens, biotin, avidin, lectins, carbohydrates, nucleic acid molecules,cells, or fragments thereof, enzymes, Protein A, molecularly-imprintedpolymers, or receptors, such as those naturally present on the surfacesof cells.

In addition, either the fixed binding member or the mobile bindingmember may be associated with a signalling substance. Examples ofsignalling substances include colored particles such as colloidal metals(e.g., colloidal gold), carbon, silica, and latex; enzymes such ashorseradish peroxidase and alkaline phosphatase; fluorophores such asfluorescein and rhodamine; liposomes; chemiluminescors; andchromophores. As indicated by the preceding list of examples, thesignalling substance may be a visible substance, such as a colored latexbead, or it may participate in a reaction by which a colored product isproduced. The reaction product may be visible when viewed with the nakedeye, or may be apparent, for example, when exposed to a specializedlight source, such as ultraviolet light. Although it is expected thatviewing the test zone (either directly or indirectly) will be theprimary way in which the test result is obtained, other methods, forexample where the analyte is associated with a radioactive substancethat is detected by subsequent exposure to X-ray film, are alsoconsidered within the scope of the invention.

The concentration of analyte in the sample, which interacts with thebinding member, will be indicated by how much of the binding member, orthe signalling substance, subsequently becomes associated with the testzone. A reaction within the test zone, that produces a measurablereaction product, indicates the analyte concentration.

Also featured in the invention are devices for determining theconcentration of an analyte in a sample. These devices consist of adefined sample application region, a defined diluent application region,and a test zone. At least one of the sample- and diluent applicationregions will have a varying width, and they will be arranged so thatthey may be brought into contact with each other. A detectable signalthat indicates the concentration of the analyte is produced in the testzone.

By "wedge-shaped" is meant any shape, which can be drawn by joiningthree or more lines, one end of which is an acute-angled edge formed bytwo converging planes. The wedge-shaped pad or wedge-shaped chamber usedin the invention will taper from a relatively large edge, or "base", toa relatively small edge, or "tip". Typically, the tip of the sampleapplication pad or chamber will be adjacent to the base of the diluentapplication pad or chamber, and vice-versa. When arranged in this waythe diluent pad or chamber is said to be "complementary" to the samplepad or chamber. The sample and diluent application pads or chambers mayor may not be identical in size or in every dimension. Furthermore,either the sample application pad, the diluent application pad, or both,may have either smooth edges or contours or one or more stepped edges orcontours.

The sample application pad or chamber and the diluent application pad orchamber may be separated by any space or physical barrier thateffectively prevents contact between the sample and diluent, and that,when dislocated, allows an analyte gradient to be established. Anexample of such a physical barrier is a strip of hydrophobicpolystyrene. Alternative means of bringing the sample into operablecontact with the diluent are described and illustrated below.

Optionally, the devices of the invention may include an indicator zonethat may be positioned either between the analyte gradient and the testzone (i.e., downstream of the sample and diluent application pads orchambers and upstream of the test zone) or downstream of the all ofthese elements (i.e., downstream of the sample application pad orchamber, the diluent application pad or chamber, and the test zone). Inthe later instance, the device may be called, as illustrated in Example3, a gradient reverse LFD.

Optionally, the devices of the invention may include a control zone,wherein a detectable signal that is independent of the analyte isproduced. Thus, the control zone may serve as an indication that thedevice is functioning properly. For example, the control zone maycontain a substance that specifically binds or otherwise specificallyinteracts with the binding member or the signalling substance that movesdownstream from the indicator zone. Typically, the control zone will bepositioned in the vicinity of the test zone.

Optionally, the devices of the invention may include one or moreabsorbent pads that are positioned to facilitate the lateral flow of theanalyte gradient through the indicator zone, the test zone, or thecontrol zone.

Optionally, the devices of the invention may include reservoirs thateither contain buffers, which may be used: (1) as a diluent, (2) toadjust the pH of any liquid within the device, or (3) to wash awayunwanted reaction products. These reservoirs may also contain reagentsthat may be necessary for detecting the analyte gradient. Reservoirscontaining buffers or reagents could be positioned next to the zonewhere their contents would be applied, and could be broken by meansincluded within the device. For example, applying pressure to aparticular point may cause pins to be pushed through a sealed bag sothat it releases its contents (U.S. Pat. No. 5,356,785). The devices mayalso contain one or more reservoirs that are of sufficient size andshape to absorb all or substantially all of the liquid that would beapplied to or released within the device in the course of performing anassay. These reservoirs may be empty or filled with an absorbentmaterial. Alternatively, excess liquids may be emptied from the devicethrough a drain.

In another aspect, the devices of the invention may be packaged togetherin a kit with any or all of the diluents or reagents needed to detect agiven analyte.

By "analyte" is meant the molecule to be assayed or an analogue orderivative thereof. Analogues or derivatives may be used when theyparticipate in an assay, as one member of a binding pair, in a mannerthat is substantially equivalent to that of the analyte itself.

The term "operable contact" is meant to define direct or indirectcontact between two solid components in any way that allows an aqueoussolution to flow in a substantially uninterrupted manner from one of thecomponents to the other component.

Various embodiments of the invention may have one or more of thefollowing advantages. The invention described herein provides a simple,rapid, and effective way to perform quantitative analyses using aportable and disposable test device. This device, the gradient LFD, canperform these analyses over a dynamic range, which can be adjusted to beas narrow or as broad as necessary. The invention establishes a spatialgradient of analyte concentration that may be simultaneously assayed anddetected along a test zone. The present invention can provide anindication of analyte concentration without the use of instrumentation.

Other features and advantages of the invention will be apparent from thefollowing description and from the claims.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of an example of a gradient lateral flowdevice, as described in Example 1. The upper housing has been removed.

FIG. 2 is an illustration of the device shown in FIG. 1, with the upperhousing, as it would appear following a sandwich-type assay, such as theassay described in Example 1.

FIG. 3 is an illustration of the device shown in FIG. 1, with the upperhousing, as it would appear following a competitive-type assay, such asthe assay described in Example 2.

FIG. 4 is an illustration of the upper surface of a gradient lateralflow device in which the viewing window is divided.

FIG. 5 is an illustration of an example of a gradient reverse lateralflow device, as described in Example 3.

FIG. 6 is an illustration of an example of a gradient lateral flowdevice that does not contain an indicator zone. The upper housing hasbeen removed.

FIG. 7 is an illustration of an example of a gradient lateral flowdevice that could be used to establish a non-linear analyte gradient.

FIG. 8 is an illustration of an example of a gradient lateral flowdevice that could be used to establish a stepped analyte gradient.

FIG. 9 is an illustration of an example of a gradient lateral flowdevice, showing the side and top housing.

FIG. 10 is a line graph depicting exemplary dose-response curves for asandwich immunoassay (interpreted from the left-hand Y axis) or acompetitive immunoassay (interpreted from the right-hand Y axis) of theinvention.

FIG. 11 is an illustration of an example of a gradient lateral flowdevice in which various pads are brought into contact by exertingpressure against the collapsable structures shown along each side.

FIG. 12 is a schematic diagram illustrating a gradient lateral flowdevice that has been configured to produce a detectable signal above 10ng/ml of analyte. The vertical numerical display reflects theconcentration of the analyte at the gradient front.

DETAILED DESCRIPTION

Before describing particular methods and devices of the invention, thegeneral features of the invention will be described. These methods anddevices provide the means to establish a liquid front, along which theconcentration of an analyte is graded, and to move that front across oneor more zones, as illustrated below. As a result, the concentration ofany given analyte in any given sample can be determined.

Establishment of an Analyte Gradient

An important feature of the invention is the creation of a liquid front,along which the concentration of the analyte being measured is graded.The analyte gradient can be established, for example, as follows. Asample, which may contain the analyte of interest, is applied to a firstpad or chamber, and a diluent, which may be any liquid that does notchemically, or otherwise specifically react with the sample, is appliedto a second pad or chamber. The sample application pad or chamber andthe diluent application pad or chamber are then brought into operablecontact with one another.

The sample and diluent application pads may be any material that iscapable of absorbing liquid. These materials include, but are notlimited to, high density (or ultra high molecular weight) polyethylene(for example, that manufactured by Porex Technologies Corp., Fairburn,Ga.), olefin or thermoplastic materials, (e.g., polyvinyl chloride,polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride),polyamide, polycarbonate, polystyrene, paper, nitrocellulose, glassfiber, polyester, and nylon.

The sample and diluent may be brought into contact, thereby establishingthe analyte gradient, in any of a number of ways. For example, the padscan be brought together by pressing on the housing at a strategic pointthat is designed to collapse under pressure and thus bring these twopads into contact. Alternatively, the device may consist of two surfacesthat are joined along one edge, for example, by a hinge. The sample maythen be placed on a pad or in a chamber on one surface, e.g., theleft-hand surface, and the diluent may be placed in a complementaryposition on the facing (e.g., the right-hand) surface. To establish thegradient, the opposing surfaces are brought into contact, in much thesame way as opposing pages of a book are brought into contact when anopen book is closed (FIG. 5; see also WO 95/16207 and WO 95/16208).Another approach entails separating the pads or chambers with ahydrophobic strip or other barrier that effectively separates the samplefrom the diluent. To establish the gradient, the strip or barrier isremoved or slid into another position to allow contact between thesample and diluent. In any event, the sample and diluent are not broughtinto contact until they have been applied to their respective pads orchambers. Any means by which the sample and diluent are initiallyseparated and subsequently brought together to form a concentrationgradient are considered within the scope of the invention.

Following application of the sample and diluent, and establishment of ananalyte gradient, flow through the device is achieved by the intrinsicproperties of the materials therein. For example, the property may beliquid transfer by capillary action or another mechanism.

The shape of the pads and their orientation are importantconsiderations. If using two smooth-edged, wedge-shaped pads, they arebrought together so that the tip of the first wedge is adjacent to thebase of the second wedge, and vice-versa. As a consequence, the analyteat the tip of the sample pad will become the most dilute (throughcontact with the large volume of diluent at the base of the diluent pad)and analyte at the base of the sample pad (through contact with thesmall volume of diluent at the tip of the pad) will become the leastdilute. Between these two points, the concentration of the analyte willbe graded. If the analyte gradient is established with two smooth-edgedpads, a smoothly graded concentration gradient will be produced. Whenthis gradient is subsequently detected, as described below, the testline that indicates the concentration of analyte will come to an end.

Alternatively, the wedge-shaped pads may have complementary steppedsurfaces. In this instance, the concentration gradient that isestablished by bringing these two pads into contact would also bestepped. The test line that will ultimately be formed to reflect astepped analyte gradient will end more abruptly than a test line thatreflects a smooth analyte gradient. Establishing a stepped analytegradient would be advantageous when it is necessary to determine only ifthe concentration of an analyte falls within a particular range: asignal would be apparent at one concentration and absent at the nexthighest (or lowest) concentration. A stepped analyte gradient is lesssubjective than a smooth analyte gradient because it is not necessaryfor the person performing the assay to judge exactly where a taperedline ends.

The analyte gradient can be steep or shallow. A steep gradient wouldexhibit a greater difference in the concentration of analyte from oneend of the gradient front to the other than would a shallow gradient.These gradients are created and can be altered by changing the lengthand shape (i.e. the angle) of the wedge-shaped sample and diluent pads.For example, long pads that are cut along one edge at a 15° angle wouldproduce a shallower gradient that short pads that are cut along one edgeat a 45° angle.

The size and shape of the sample and diluent application pads orchambers will determine both the depth of the concentration gradient(i.e., the magnitude of the dilution; an analyte may be diluted e.g.,1-fold or 100-fold from one end of the gradient to the other), and thedynamic concentration range over which dilution will occur. The spatialorientation of the analyte gradient, whether smooth or stepped, issubsequently maintained as the analyte participates, as the first memberof a binding pair, with the second member of a binding pair in theindicator zone or the test zone.

In general, assays are imprecise when a large change in analyteconcentration produces only a small change in the signal (curve "b" onFIG. 10). FIG. 10 illustrates how a gradient LFD can be configured toextend the dynamic range of an assay and, at the same time, take fulladvantage of the desirable precision characteristics of assays thatnormally have steep or threshold dose-response characteristics (e.g.,curve "a" in FIG. 10).

FIG. 10 shows two dose response curves ("a" and "b"). Interpreted fromthe left-hand Y axis, the curves illustrate example dose-responses forsandwich (e.g., immunometric) assays, which typically display anincrease in signal intensity as analyte concentration increases.Interpreted from the right-hand Y axis, the curves illustrate exampledose-responses for competitive assay, which typically result in adecrease in signal intensity as analyte concentration increases. Curve"a" shows a steep dose response, which is normally associated with highprecision and narrow dynamic range, while curve "b" shows a shallow doseresponse, which is normally associated with lower precision but a broaddynamic range. The loss of precision in moving from curve "a" to curve"b" can be seen by extrapolating identical variations in signalintensity (from the Y axis) into the corresponding concentrationdifferences along the X axis using each of the two curves. In mostanalytical situations, it would be advantageous to maintain theprecision afforded by curve "a" but at the same time have the ability toguantitate over a wide dynamic range, as shown in curve "b". Toaccomplish this in practice, a series of sample dilutions could beanalyzed using the assay of curve "a" with the results subsequentlycorrected to account for the dilutions used.

Lateral flow assays provide qualitative information, i.e., whether ananalyte is above or below a defined concentration. This qualitativeresult is visually interpreted by either the presence or absence of atest line on the device following the analysis. FIG. 10 illustrates theportion of the dose response curves where a signal either begins tobecome visible or where the signal becomes invisible ("whited out"). Fora sandwich assay, the sensitivity would be defined as the point at whicha visible signal can first be observed, while for a competitive assays,the sensitivity is defined as the point at which a visible signal can nolonger be seen. An assay exhibiting curve "a" provides a sharper andmore precise interpretation threshold between positive and negative thancurve "b" and thus provides a more desirable threshold assay. Thesensitivity of particular assays is a function of the relative affinityand concentration of the various reagents and the times during whichparticular reagents and analyte are in contact with each other. Thoseskilled in the art are familiar with methods for optimizing andcharacterizing the sensitivity and dose-response characteristics ofconventional assays used in lateral flow devices. A sandwich assayhaving the dose response characteristics of a curve "a" could be used toproduce a conventional lateral flow device that has a sensitivity of 10ng/ml. Thus, if the sample contains analyte at 10 ng/ml or greater,there will be sufficient analyte to react with the fixed test line andto capture sufficient signaling substance to result in a detectablesignal. The detectable signal will be of the same intensity across thewidth of the test line since the analyte normally travels across thewidth of the test line in a uniform concentration. If the samplecontains analyte at 100 ng/ml, a signal would develop at the test lineand the results would still be interpreted the same, analyte isdetected. By performing a series of dilutions on this sample one coulddetermine that it takes a tenfold dilution to reach the sensitivitylimit of the LFD and therefore the sample contains about 100 ng/ml ofanalyte.

The gradient lateral flow device provides a means of using curve "a"while at the same time creating a gradient of sample dilution across apre-defined dynamic range (defined by the size and shape of thecomplementary sample and dilution pads or chambers). Thus, for a samplecontaining an analyte at 100 ng/ml, the test line will remain visibleacross a distance of the analyte gradient that is equivalent to a10-fold drop in concentration (FIG. 12). Beyond that distance, theanalyte concentration will be below the threshold of the assay andtherefore, no test line will be detected. The higher the analyteconcentration, the further into the gradient the analyte must move to bediluted to the detection limit of the assay, and therefore the greaterthe length of detectable test line following the assay. For competitiveassays, the same situation applies, except that the detectable line willbe at its longest for no or low analyte concentration and increasingconcentrations of analyte in the sample will result in decreasinglengths of detectable test line following the assay.

The Indicator Zone

The analyte, present along the concentration gradient described above,will move downstream as a front and may next contact the indicator zone,which contains a binding member. The binding member in the indicatorzone interacts with the analyte in the sample and becomes mobile uponcontact with the moving analyte gradient front. The length of theindicator zone will be approximately the same as the length of theanalyte gradient front, and the two will be parallel to one another. Theindicator zone may also contain an appropriate signalling substance suchcolored latex beads, or silica, or liposomes that have encapsulatedchemiluminescors (e.g., luciferin) or chromophores (e.g., dyes, orpigments). The signalling substance may also consist of a colloid systemcontaining, for example, colloidal carbon or a dispersion of a metalsuch as gold or silver, which may be associated with the mobile bindingmember. Alternatively, the binding member may be associated with asignalling substance that is not particulate such as a dye, afluorophore, enzyme, or a chemiluminescor.

Examples of binding pairs that may form in the indicator zone (either ofwhich could be the analyte or the binding member) are: antigen-antibody,antigen-antibody fragment, antibody-hapten, antibody-cell, antibody-cellfragment, nucleic acid-nucleic acid, receptor-ligand, enzyme-substrate,biotin-avidin, lectin-carbohydrate, Protein A-immunoglobulin, andmolecularly imprinted polymer (MIP)-imprinted compound.

Because of the properties of the materials within the gradient LFD, theanalyte will move through the indicator zone, mobilizing the bindingmember therein, and presenting it for subsequent reaction (ornon-reaction) with the fixed binding member present in the test zone.

The Test Zone

The analyte gradient may move into the indicator zone, as describedabove, and then downstream to the test zone. Alternatively, the analytegradient may flow directly into the test zone. In the later instance,the concentration of the analyte in the sample may be determinedimmediately, or after subsequent contact between the test zone and theindicator zone. In any event, the test zone will contain a fixed bindingmember that reacts either with the analyte, or with the mobile bindingmember that has been carried to the test zone from the indicator zone.

The gradient LFD may be used to carry out assays analogous to thosereferred to as "competitive" or "sandwich" (immunometric) typeimmunoassays. "Competitive" assays are those in which a labeled antigenor antibody competes with the antigen or antibody being assayed for animmunological binding site on a solid phase, for example, a bead or pad.The "sandwich" assay differs from this in that the antibody or antigenbeing assayed is sandwiched between a solid surface treated with onemember of a complementary binding pair and either the same or adifferent complementary binding member that has been coupled to adetectable label.

The Gradient LFD can be used to perform both competitive and sandwichtype assays. Typically, large molecules (those greater thanapproximately 5000 Da) will be assayed with the gradient LFD using asandwich type configuration. This leads to the presence of a test linewhen the analyte concentration is above a specified thresholdconcentration. For lower molecular weight compounds, a competitive typeconfiguration is used, which may result in an inhibition or "white out"of a test line when the analyte concentration is above a specifiedthreshold. However, certain competitive assays have been developed inwhich the test line, rather than being inhibited, becomes visible (see,e.g., U.S. Pat. No. 5,527,686). In either event, interpretation of theconcentration of analyte will be made by inspecting the line anddetermining at what point it becomes apparent or disappears.

The detectable test line forms in the test zone as the result of thespecific association between the fixed binding member in the test zoneand either the analyte, or the mobile binding member that has interactedwith the analyte in the indicator zone. The binding member in the testzone may consist of any of the agents specified above as bindingmembers. Similarly, it may be associated with the same signallingsubstances described above.

The Control Zone

A control zone that contains a line, or some other configuration, suchas a spot, that becomes detectable in a manner that is independent ofthe analyte gradient can also be included in the device. Preferably, thecontrol zone is in the vicinity of the test zone and consists of animmobilized binding member that reacts with some portion of a bindingmember, or signalling substance, from the indicator zone.

The test zone and the control zone may be covered with a clear ortranslucent cover to facilitate visualizing the signal generated. Thecover may be uniform, or it may be punctuated by, for example, lines orbars that facilitate determining whether the concentration of theanalyte falls within a particular range.

EXAMPLE I

There is described below the structure and operation of a particulargradient LFD of the invention, which is used to conduct a sandwich-typeassay.

Structure

FIG. 1 is an illustration of a gradient LFD, showing a top view of thedevice from which the upper housing has been removed. This deviceincludes a wedge-shaped pad of absorbent paper (1; e.g., absorbent grade470 paper from Schleicher and Schuell, Keene, N.H.) to which diluent isapplied, and a complementary wedge-shaped pad (2), of the same orsimilar material, to which sample is applied. The diluent pad (1) isseparated from the sample pad (2) by a strip of hydrophobic polystyrene(8; Filter, Flow, and Seal Inc., Claymont, Del.) that may be displaced.The indicator zone consists of a pad of polyester (4; AhlstromFiltration, Mt. Holly Springs, Pa.) that is the same width as the sampleapplication pad (2), from which it is separated by a second strip ofhydrophobic polystyrene (3) that may also be displaced. The hydrophobicpolystyrene strip (8), which separates the diluent pad (1) from thesample pad (2), projects from the housing as a tab, as does thehydrophobic polystyrene strip (3), which separates the sample pad (2)from the indicator zone (4). This arrangement allows the strips to bemanually displaced by pulling on the exposed tabs. The indicator pad (4)is in direct contact with and slightly overlapping, a piece ofnitrocellulose membrane (5; e.g., 15 μm fast flow nitrocellulose;Millipore Corporation, Bedford, Mass.) that contains two zones ofimmobilized reagents: the test zone (9) and the optional control zone(10). Nitrocellulose membrane 5 is in direct contact with, and slightlyoverlapped by, an absorbent pad (6) that serves as a sink for excesssample and diluent that has been wicked from pads 1 and 2 through pads 4and 5. The absorbent elements described are oriented and fixed in placealong a strip of adhesive-backed plastic (7).

The upper surface of the housing (17) of the gradient LFD is shown inFIG. 2. Sample is introduced through a sample port (11) and diluent isintroduced through a diluent port (12). The housing contains a window(16) that exposes the nitrocellulose membrane (5) for viewing. A scale(13) can be employed as an aid to quantifying the concentration ofanalyte. Following analysis of a sample, a visual reaction product willbe evident in the test zone (9) and in the control zone (10).

To manufacture the device, a 4 cm×8 cm plastic card (7) is covered withdouble-sided adhesive tape (Adhesives Research Inc., Glen Rock, Pa.),and the various absorbent members are attached to the adhesive-coatedplastic card. The nitrocellulose (5; 4 cm×2 cm), whose left 4 cm side islocated parallel to and 4 cm from the left edge of the plastic card, isadhered first. The indicator zone (4; 4 cm×1 cm) is adhered to the leftof the nitrocellulose pad (5) and overlaps it by 2 mm. The sampleapplication pad (2; a right triangular pad 4 cm×2 cm×4.47 cm) is adheredto the left of (4) and overlaps it by 2 mm along its 4 cm edge. Thestrip of hydrophobic polystyrene (3) is placed between the overlap ofthe sample application pad (2) and (4) to prevent contact between thepads. The diluent application pad (1; a right triangular pad 4 cm×2cm×4.47 cm) is located to the left of (2) and overlaps it by 2 mm alongthe 4.47 cm edge. The strip of hydrophobic polystyrene (8) is placedbetween the diluent application pad (1) and the sample application pad(2) to prevent contact of the pads. The sink pad (6; 4 cm×2 cm) isadhered to the right of the nitrocellulose pad (5) and overlaps it by 2mm along its 4 cm edge. The card assembly described is then placed intoa plastic housing (17) that: positions the sample port (11) over thesample application pad (2); positions the diluent application port (12)over the diluent application pad (1); and positions the viewing window(16) over the test zone (9) and control zone (10) of the nitrocellulosemembrane (5).

Operation

The device illustrated in FIG. 1 may be used to quantify a humanimmunoglobulin molecule, such as an antibody of the IgG type. To preparethe device for this assay, the pads within the various zones are treatedas follows.

The absorbent pad within the indicator zone (4) is pre-blocked for 30minutes with a solution consisting of 50 mM phosphate, 0.5% PVA, 0.5%BSA, and 0.1% Triton X-100 (Ph 7.4). Goat anti-human IgG colloidal goldconjugate (Sigma Chemical Co., St. Louis, Mo.) having an optical densityat 540 nm of 15 absorbance units is applied across the entire 4 cm widthof the indicator zone pad 4 in a line 2 mm wide, and at a rate of 4 μlgold reagent per linear cm using a TLC sprayer (Camag, Wilmington,N.C.). The 2 mm wide line runs perpendicular to the center of the 1 cmedge of the indicator zone pad (4).

The sample application pad 2 is pre-blocked with a solution consistingof 0.01M sodium borate and 0.1% Triton-X-100 (pH 8.6). The sampleapplication pad (2) is then dried overnight at 37° C.

The reagents that are found at the test zone (9) and control zone (10)can be applied to 15 μm fast flow nitrocellulose (5; 4 cm×2 cm) with aTLC sprayer (Camag, Wilmington, N.C.). The reagent applied to the testzone is goat anti-human IgG (Sigma Chemical Co., St. Louis, Mo.) whichis diluted in phosphate buffer and applied across the entire 4 cm widthof the nitrocellulose in a line 1 mm wide and at a concentration of 2 μgof antibody per linear cm. The 1 mm wide line runs perpendicular to the2 cm edge of the nitrocellulose (5) at a distance of 8 mm from the 4 cmwide edge that contacts the indicator zone pad (4). The reagent appliedto the control zone (10) is rabbit anti-goat IgG (Sigma Chemical Co.,St. Louis, Mo.) which is diluted in phosphate buffer and applied acrossthe entire 4 cm width of the nitrocellulose in a line 1 mm wide and at aconcentration of 4 μg of antibody per linear cm. The 1 mm wide line runsperpendicular to the 2 cm edge of the nitrocellulose (5) element at adistance of 12 mm from the 4 cm wide edge that contacts the indicatorzone pad (4). The nitrocellulose (5) is then dried overnight at 37° C.

The concentration of human IgG in a serum sample is quantified by firstadding 300 μl of serum to the sample application port and 300 μl ofphosphate buffer to the diluent application port. To establish ananalyte gradient, strip 8 is withdrawn from the side of the housing,allowing the diluent application pad (1) to contact the sampleapplication pad (2). To initiate the assay, strip 3 is withdrawn fromdevice allowing sample application pad (2) to contact the indicator zone(4). Thus, initiating the flow of the graded analyte front laterallythrough the indicator zone (4), the test zone (9), the control zone(10), and the sink (6). As the liquid front moves through the indicatorzone, the goat anti-human IgG-colloidal gold is rehydrated and carriedlaterally toward the test zone (9). As this occurs, the human IgG in theserum sample binds to the anti-human IgG colloidal gold conjugate. Theconcentration of colloidal gold across the gradient front remainsconstant. However, the amount of human IgG that is bound to theanti-human IgG on the gold particles will be directly proportional toits concentration at any given position along the gradient front. As thegold particles pass across the immobilized anti-human IgG located in thetest zone (9), the antibody-gold conjugate particles that have boundhuman IgG will be captured and concentrated. A visual line will form inthe test zone (9) that is proportional in length to the concentration ofhuman IgG in the serum sample. The concentration of IgG is interpretedfrom a reference scale (13), which is placed on the housing, at the edgeof the viewing window. After passing the test zone, the gold frontcontinues to move laterally across the control zone, which containsimmobilized rabbit anti-goat IgG. As the gold passes across theanti-goat IgG located in the control zone (10), gold particles, whichall carry the goat anti-human IgG, are captured and concentrated. Avisual line forms across the entire control zone (10), indicating thatthe assay has functioned properly. The excess liquid and reagents willcontinue to move laterally across the device and collect in the sink(6).

When used as described above, the gradient LFD detects antibodies of theIgG type in the range of 25-25,000 μg/ml.

EXAMPLE 2

There is described below the structure and operation of a particulargradient LFD of the invention, which is used to conduct acompetitive-type assay.

Structure

The structure of the device described here, which is used to conduct acompetitive-type assay, is identical to the device described in Example1(and shown in FIG. 1). However, with the device used in this instance,the disappearance (rather than the appearance) of the test lineindicates the concentration of analyte in the sample (see FIG. 3).

Operation

The gradient LFD may be used to quantify the concentration of theherbicide atrazine in a water sample. In this instance, the absorbentpad within the indicator zone (4) is pre-blocked for 30 minutes with asolution consisting of 50 mM phosphate, 0.5% PVA, 0.5% BSA, and 0.1%(v/v) Triton X-100 (pH 7.4). The pad is then dried overnight at 37° C.The mouse anti-atrazine IgG-colloidal gold conjugate (Biostride Inc.,Palo Alto, Calif.) having an optical density at 540 nm of 15 absorbanceunits is applied across the entire 4 cm width of the indicator zone pad(4) in a line 2 mm wide, and at a rate of 4 μl gold reagent per linearcm using a TLC sprayer (Camag, Wilmington, N.C.). The 2 mm wide lineruns perpendicular to the center of the 1 cm edge of the indicator zonepad (4).

The sample application pad (2) is pre-blocked with a solution consistingof 0.01M sodium borate and 0.1% Triton-X-100 (pH 8.6). The sampleapplication pad (2) is then dried overnight at 37° C.

The reagents that are found at the test zone (9) and control zone (10)can be applied to 15 μm fast flow nitrocellulose (5; 4 cm×2 cm) with aTLC sprayer (Camag, Wilmington, N.C.). The reagent applied to the testzone is atrazine-BSA (Biostride Inc., Palo Alto, Calif.) which isdiluted in phosphate buffer and applied across the entire 4 cm width ofthe nitrocellulose in a line 1 mm wide and at a concentration of 2 μg ofantibody per linear cm. The 1 mm wide line runs perpendicular to the 2cm edge of the nitrocellulose (5) at a distance of 8 mm from the 4 cmwide edge that contacts the indicator zone pad (4). The reagent appliedto the control zone (10) is rabbit anti-mouse IgG (Sigma Chemical Co.,St. Louis, Mo.) which is diluted in phosphate buffer and applied acrossthe entire 4 cm width of the nitrocellulose in a line 1 mm wide and at aconcentration of 4 μg of antibody per linear cm. The 1 mm wide line runsperpendicular to the 2 cm edge of the nitrocellulose (5) at a distanceof 12 mm from the 4 cm wide edge that contacts the indicator zone pad(4). The nitrocellulose (5) is then dried overnight at 37° C.

The concentration of atrazine in the water sample is quantified byadding 300 μl of the water sample to the sample application port and 300μl of phosphate buffer to the diluent application port. To establish ananalyte gradient, strip 8 is withdrawn from the side of the housing,allowing the diluent application pad (1) to contact the sampleapplication pad (2). To initiate the assay, strip 3 is withdrawn fromdevice allowing sample application pad (2) to contact the indicator zone(4). Thus, initiating the flow of the graded analyte front laterallythrough the indicator zone (4), the test zone (9), the control zone(10), and the sink (6). As the liquid front moves through the indicatorzone, the mouse anti-atrazine IgG-colloidal gold is rehydrated andcarried laterally toward the test zone (9). The atrazine in the watersample binds to the anti-atrazine IgG-colloidal gold. The concentrationof colloidal gold across the gradient front remains constant. However,the amount of atrazine that is bound to the anti-atrazine IgG on thegold particles will be directly proportional to its concentration at anygiven position along the gradient front. As the gold particles passacross the atrazine-BSA located in the test zone (9), gold particlescarrying the atrazine blocked antibody will pass across the test zonewithout binding to the immobilized atrazine-BSA conjugate. A visual linewill form in the test zone (9) that is inversely proportional in lengthto the concentration of atrazine in the water sample. The concentrationof atrazine is interpreted from a reference scale (13), which is placedon the housing, at the edge of the viewing window. After passing thetest zone, the gold front continues to move laterally across the controlzone, which contains immobilized rabbit anti-mouse IgG. As the goldpasses across the anti-mouse IgG located in the control zone (10), goldparticles, which all carry the mouse anti-atrazine IgG, are captured andconcentrated. A visual line forms across the entire control zone (10),indicating that the assay has functioned properly. The excess liquid andreagents will continue to move laterally across the device and collectin the sink (6).

When used as described above, the gradient LFD detects atrazine in therange of 5-500 ng/ml.

EXAMPLE 3

There is described below the structure and operation of a particulargradient LFD of the invention, which functions as a gradient reverseflow device (GRFD).

Structure

FIG. 5 shows a view of a Gradient Reverse Flow Device (GRFD). Thisdevice includes a right wedge-shaped pad (4 cm×2 cm×4.47 cm) ofabsorbent material (27) such as absorbent paper (e.g., grade 470 paper;Schleicher and Schuell, Keene, N.H.) which serves as the diluentapplication pad. Similarly, the sample application pad (25) is awedge-shaped pad (4 cm×2 cm×4.47 cm) of absorbent material such asabsorbent paper (e.g., grade 470 paper; Schleicher and Schuell, Keene,N.H.) which serves as the sample application pad. The sample applicationpad (25) is attached to a non-absorbent page of coated cardboard (26; 3cm×5 cm) that is attached through a hinge (23) to the opposing page(28). The sample application pad (25) can be brought into contact withboth the diluent application pad (27) and the nitrocellulose membrane(29; 4 cm×2 cm) by first removing a strip of protective paper (33), thusexposing the underlying adhesive strip (24). The bottom of the left-handpage (26) may then fold down over the right-hand page (28) so that thebottom left-hand page (26) is held in place by the adhesive strip (24)and the diluent application pad (27), the sample application pad (25),and the nitrocellulose pad (29), are positioned so that their edges areall in contact and overlapping by approximately 2 mm. The indicator zone(34; 4 cm×1.5 cm) is comprised of a rectangular piece of polyester(Ahlstrom Filtration, Mt. Holly Springs, Pa.) and is positioned on thetop of the left-hand page (36; 4.5 cm×5 cm) such that when the top ofthe left-hand page (36) and the right-hand page (28) are broughttogether, the indicator zone (34) will be in direct contact with andoverlapping, by approximately 2 mm, the nitrocellulose pad (29;Millipore Corporation, Bedford, Mass.). Also positioned on the topleft-hand page (36) is an absorbent pad (35; 4 cm×0.5 cm) which can bebrought into direct contact with the nitrocellulose pad (29), byadhering the edge of the top left-hand page (36) with the adhesive strip(24) on the right-hand page (28). The absorbent pad (35) will completelyoverlap the nitrocellulose pad (29). A window (22) is positioned on thetop left-hand page (36) such that the line that will develop in the testzone (31) and the line that will develop in the control zone (30) can beviewed following the assay. A reverse line (32) printed on theright-hand page (28) provides a reference point indicating that acertain volume of liquid has passed across the test line and that theflow of liquid should be reversed by folding the top left-hand page (36)onto the right-hand page (28), as previously described. Determining thevolume of liquid that will optimally pass the reverse line is determinedempirically for each assay.

Operation

The device illustrated in FIG. 5 may be used to quantify a humanimmunoglobulin molecule, such as an antibody of the IgG type. To preparethe device for this assay the pads within the various zones are treatedas follows.

The indicator zone (34) is pre-blocked for 30 minutes with a solutionconsisting of 50 mM phosphate, 0.5% PVA, 0.5% BSA, and 0.1% Triton X-100(pH 7.4). A goat anti-human IgG colloidal gold conjugate (Sigma ChemicalCo., St. Louis, Mo.) having an optical density at 540 nm of 15absorbance units is applied across the entire 4 cm width of theindicator zone (34) in a line 2 mm wide and at a rate of 4 μl of goldreagent per linear cm using a TLC sprayer (Camag, Wilmington, N.C.). The2 mm wide line runs perpendicular to the 1.5 cm edge of the indicatorzone (34) centered on a point 3 mm from the edge of the pad that is inopposable contact with the nitrocellulose pad (29).

The sample application pad (25) is pre-blocked with a solutionconsisting of 0.01M sodium borate and 0.1% Triton X-100 (pH 8.6). Thesample application pad is then dried overnight at 37° C.

The reagents within the test zone (31) and the control zone (30) can beapplied to 15 μm fast flow nitrocellulose (29; 4 cm×2.5 cm) with a TLCsprayer (Camag, Wilmington, N.C.). The reagent applied to the test zone(31) to create the test line is goat anti-human IgG (Sigma Chemical Co.,St. Louis, Mo.). The antibody is diluted in phosphate buffer and appliedacross the entire 4 cm width of the zone in a line that is 1 mm wide.The concentration of this antibody is 2 μg per linear cm. The test lineruns perpendicular to the 2.5 cm edge of nitrocellulose pad (29) at adistance of 10 mm from the 4 cm wide edge that is in opposable contactwith the sample application pad (25). The reagent applied to the controlzone (30), rabbit anti-goat IgG (Sigma Chemical Co., St. Louis, Mo.), isdiluted in phosphate buffer and applied across the entire 4 cm width ofthe zone in a line that is 1 mm wide. The concentration of this antibodyis also 2 μg per linear cm. Similarly, the 1 mm wide control line runsperpendicular to the 2.5 cm edge of the nitrocellulose pad (29), but ata distance of 14 mm from the 4 cm wide edge that is in opposable contactwith the sample application pad (25). The test zone and the controlzones are dried overnight at 37° C.

The concentration of human IgG in a serum sample is quantified by firstadding 300 μl of serum to the sample application pad (25) and 300 μl ofphosphate buffer to the diluent application pad (27). To establish ananalyte gradient and to initiate flow of the gradient through thenitrocellulose membrane, sample application pad (25) is brought intocontact with diluent application pad (27) and the nitrocellulose pad(29). This is accomplished by exposing an adhesive strip (24) byremoving an overlying protective paper (33) and then folding andadhering the bottom half of the left-hand page (26) to the right-handpage (28) along their outer edges. The dry reagent in the indicator zone(34) is then rehydrated by adding 200 μl of phosphate buffer.

As the liquid front of the analyte gradient passes across the controlzone (30) and test zone (31), any human IgG in the sample will becaptured by the anti-human IgG antibody, fixed at the test zone (31), inan amount that is directly proportional to its position across thegradient front. When the liquid gradient front reaches the reverse line(32), the indicator zone (34) is brought into contact with thenitrocellulose pad (29) and the absorbent pad (35) is brought intocontact with the nitrocellulose membrane (29) by folding the top half ofthe left-hand page (36) onto the right-hand page (28) and adhering thetwo pages together at their edges along the adhesive strip (24). Thisinitiates reverse flow whereby the goat anti-human IgG-colloidal gold inthe indicator zone (34) moves through the nitrocellulose pad (29),across the test zone (31) and the control zone (30) and into theabsorbent pad (35). As the goat anti-human IgG-colloidal gold passesacross the test zone (31) it will bind to any human IgG that hadpreviously been captured and concentrated by the goat anti-human IgGfixed at the test zone. A visual line will form that is proportional inlength to the concentration of human IgG in the serum sample. Theconcentration of IgG is interpreted from a reference scale, which isplaced along the outside edge of the viewing window (22). After passingthe test zone, the gold front continues to move laterally across thecontrol zone (30) which contains immobilized rabbit anti-goat IgG. Asthe gold passes across the anti-goat IgG located in the control zone,gold particles which all carry the goat anti-human IgG are captured andconcentrated. A visual line forms across the entire control zoneindicating that the assay has functioned properly. The excess liquid andreagents will continue to move through the membrane and into theabsorbent pad (35).

Other Embodiments

Still further embodiments are possible.

In FIG. 6 there is shown a gradient lateral flow device wherein theindicator zone is absent. In this device, an analyte gradient isestablished and brought directly into contact with a nitrocellulose padon which a test line and a control line have been formed.

In FIG. 7 there is shown a gradient lateral flow device wherein anon-linear analyte gradient is established. In this configuration, thecomplementary sample and diluent application pads are attached tosurfaces that can be brought into operable contact by folding themtoward one another. In FIG. 7, the sample application pad (25A) isattached to the bottom left-hand page (26) and the diluent applicationpad (27A) is attached to the right-hand page (28).

In FIG. 8 there is shown a gradient lateral flow device wherein astepped analyte gradient is established.

In FIG. 11 there is shown a gradient lateral flow device in which thematerial to which the various pads are attached (7) containsaccordion-like ridges that will collapse under pressure. The ridges thatseparate the sample application pad (2) from the diluent application pad(1), i.e. ridges (50) and (50A) will be designed to collapse first. Theridges that separate the diluent application pad (1) from the indicatorzone (4), i.e. ridges (60) and (60A) will be designed to collapsesubsequently.

Other embodiments are within the following claims.

What is claimed is:
 1. A method of determining the concentration of ananalyte in a sample, said method comprising:(a) establishing an analytegradient in a lateral flow device; (b) bringing said gradient intoliquid contact with an indicator zone containing a mobile binding memberconjugated to a signalling substance, wherein said mobile binding membereither (i) binds to said analyte or (ii) competes with said analyte forbinding to a fixed binding member contained in a test zone; and (c)bringing said indicator zone into liquid contact with said test zonecontaining said fixed binding member, wherein said fixed binding memberbinds to said analyte or said mobile binding member, and a detectablesignal that indicates the concentration of said analyte is produced. 2.A method of determining the concentration of an analyte in a sample,said method comprising:(a) establishing an analyte gradient; (b)bringing said gradient into liquid contact with a test zone containing afixed binding member wherein a detectable signal that indicates theconcentration of said analyte is produced.
 3. A method of determiningthe concentration of an analyte in a sample, said method comprising:(a)establishing an analyte gradient in a lateral flow device; (b) bringingsaid gradient into liquid contact with a test zone containing a fixedbinding member wherein said fixed binding member binds to said analyte;and (c) bringing an indicator zone into liquid contact with said testzone, wherein (i) said indicator zone contains a mobile binding memberconjugated to a signaling substance and (ii) said mobile binding memberbinds to said analyte or said fixed binding member contained in saidtest zone, and a detectable signal that indicates the concentration ofsaid analyte is produced.
 4. The method of claim 1, claim 2, or claim 3,wherein said analyte is an antibody, an antibody fragment, an enzyme, ahormone, a growth factor, a neurotransmitter, a nucleic acid, or ahapten.
 5. The method of claim 1, claim 2, or claim 3, wherein saidanalyte is a bacterial, fungal, parasitic, or viral antigen.
 6. Themethod of claim 1, claim 2,or claim 3, wherein said analyte is a drugwhich is a barbiturate, an amphetamine, methadone, morphine, cocaine,codeine, dilaudid, tetrahydrocannabinol, or diazepan.
 7. The method ofclaim 1, claim 2, or claim 3, wherein said analyte is used in themanufacture of food, industrial agents, or chemical products.
 8. Themethod of claim 1, claim 2, or claim 3, wherein said analyte is abulking agent, a vitamin, a colorant, a flavorant, an insecticide, anherbicide, a fertilizer, a surfactant, an adhesive, a resin, an organicpollutant, a hapten, or a process chemical.
 9. The method of claim 1,claim 2, or claim 3, wherein said sample is a biological fluid.
 10. Themethod of claim 9, wherein said biological fluid is whole blood, serum,plasma, cerebrospinal fluid, or urine.
 11. The method of claim 1, claim2, or claim 3, wherein said sample is an environmental sample.
 12. Themethod of claim 11, wherein said environmental sample is soil, foliage,or water.
 13. The method of claim 1, claim 2, or claim 3, wherein saidmobile binding member and said fixed binding member are selected fromthe group consisting of an antibody, an antibody fragment, a hapten, acell, a cell fragment, a nucleic acid molecule, a cell surface receptor,a lectin, a carbohydrate, avidin, biotin, Protein A, or an enzyme. 14.The method of claim 1, claim 2, or claim 3, wherein said signallingsubstance comprises a liposome, a latex bead, colored silica, colloidalmetal, a chemiluminescer, an enzyme, a fluorophore, or a chromophore.15. The method of claim 2, wherein said analyte binds to said fixedbinding member.
 16. The method of claim 1, claim 2, or claim 3, whereinsaid analyte gradient is established by:(a) applying said sample to adefined sample application region comprising an absorbent materialhaving a width which tapers from a large base to a small tip; (b)applying a diluent to a defined diluent application region comprising anabsorbent material; and (c) bringing said sample application region intocontact with said diluent application region to establish an analytegradient front.
 17. The method of claim 16, wherein said sample anddiluent application pads have smooth edges.
 18. The method of claim 16,wherein at least one of said sample and diluent application pads has atleast one stepped edge.
 19. A lateral flow device for determining theconcentration of an analyte in a sample, said device comprising:(a) adefined sample application region comprising an absorbent materialhaving a width which tapers from a large base to a small tip; and (b) adefined diluent application region comprising an absorbent material;wherein said regions are physically arranged to be brought into liquidcontact with each other to establish an analyte gradient front; and (c)a test zone, physically arranged to be brought into liquid contact withsaid analyte gradient front, wherein said test zone contains a reagentfor providing a detectable signal that indicates the concentration ofsaid analyte.
 20. The device of claim 19, wherein said reagent in saidtest zone is a fixed binding member which binds either to said analyteor a mobile binding member, said device further comprising an indicatorzone containing said mobile binding member conjugated to a signallingsubstance, wherein said mobile binding member either (i) binds to saidanalyte or (ii) competes with said analyte for binding to a fixedbinding member contained in said test zone.
 21. The device of claim 20,wherein said indicator zone is positioned downstream from said sampleand diluent application regions and upstream from said test zone. 22.The device of claim 20, wherein said indicator zone is positioneddownstream from said sample application region and said diluentapplication region, such that the indicator zone can be brought intoliquid contact with the test zone to effect reverse flow of the mobilebinding member from the indicator zone across the test zone.
 23. Thedevice of claim 20, further comprising a control zone containing asecond fixed binding member, wherein said second fixed binding memberbinds to said mobile binding member to produce a detectable signal thatis independent of said analyte.
 24. The device of claim 23, furthercomprising one or more absorbent pads that are positioned to facilitatethe lateral flow of said analyte gradient front through the indicatorzone, the test zone, or the control zone.
 25. The device of claim 19,wherein said diluent application region also has a width which tapersfrom a large base to a small tip and wherein the tip of said sampleapplication region is adjacent to the base of said diluent applicationregion, and vice-versa.
 26. The device of claim 19, wherein said sampleapplication region and said diluent application region have smoothedges.
 27. The device of claim 19, wherein at least one of said sampleand diluent application regions has at least one stepped edge.
 28. Thedevice of claim 19, further comprising a physical barrier between saidsample application region and said diluent application region, whereinsaid sample application region is brought into liquid contact with saiddiluent application region by dislocation of said physical barrier. 29.The device of claim 28, wherein said physical barrier is a strip ofhydrophobic polystyrene.
 30. The device of claim 19, wherein said sampleapplication region is brought into liquid contact with said diluentapplication region by bringing a first surface, to which said sampleapplication region is attached, into contact with a second surface, towhich said diluent application region is attached.
 31. The device ofclaim 30, wherein said first surface and said second surface areattached to one another along one edge, each of said surfaces having anopposite unattached edge, said contact being established by bringingsaid unattached edge of said first surface into contact with saidunattached edge of said second surface.
 32. The device of claim 30,wherein said first surface and said second surface are separated fromeach other by ridges which collapse and fold like an accordion underpressure and are brought into contact by applying pressure to one ormore points on said device, said pressure being sufficient to collapsesaid ridges and thereby bring said first surface into contact with saidsecond surface.
 33. The device of claim 19, further comprising a drainwhereby sample, diluent, or reagent used to detect said analyte can beremoved from said device subsequent to traversal through said device.34. The device of claim 19, further comprising reservoirs containingbuffers or reagents used to detect said analyte, wherein said reservoirsare positioned adjacent to a point of application.
 35. A kitcomprising(a) the device of claim 19, and (b) one or more diluents. 36.A kit comprising:(a) the device of claim 19, and (b) one or morereagents.
 37. A kit comprising:(a) the device of claim 19, (b) one ormore diluents, and (c) one or more reagents.